Packing material for liquid chromatography and process for producing the same

ABSTRACT

Column packing materials useful in applications such as liquid chromatography, as well as a process for producing such a packing material are described. A first aspect of the invention concerns a column packing material composed of a spherical substrate having a coating of a calcium phosphate based compound on the surface thereof. 
     A second aspect of the invention concerns a packing material comprising porous calcium phosphate based granules having open pores with an average pore size of from 0.01 to 20 μm, said granules being composed of crystalline particles with an average size of from 0.1 to 10 μm. 
     A third aspect of the invention concerns a spherical packing material for liquid chromatography comprising spherical particles of at least one material selected from the group consisting of Ca 10 (PO 4 ) 6 (OH) 2 , Ca 3 (PO 4 ) 2 , Ca 2 P 2 O 7 , Ca(PO 3 ) 2 , Ca 10 (PO 4 ) 6 F 2  and Ca 10 (PO 4 ) 6 Cl 2 , said packing material having a dense structure with a porosity of no more than 5%. 
     A fourth aspect of the invention concerns a packing for liquid chromatography comprising particles having at least on the surface thereof a fluoroapatite represented by formula (I): 
     
       
         Ca 10 (PO 4 ) 6 (OH) 2−2x F 2x   (I) 
       
     
     wherein x is a number of from about 0.1 to 1.

This is a divisional of application Ser. No. 08/029,940, filed Mar. 10,1993, now U.S. Pat. No. 5,441,635, which is a continuation ofapplication Ser. No. 07/908,740, filed Jul. 6, 1992 (now abandoned),which is a continuation of U.S. application Ser. No. 07/654,982, filedFeb. 14, 1991 (now abandoned), which is a continuation of U.S.application Ser. No. 07/461,854, filed Jan. 8, 1990 (now U.S. Pat. No.5,039,408), which is a Continuation-in-Part of U.S. application Ser. No.07/069,734, filed Jul. 6, 1987 (now abandoned), and aContinuation-in-Part of U.S. application Ser. No. 07/069,742, filed Jul.6, 1987 (now abandoned), and a Continuation-in-part of U.S. applicationSer. No. 07/216,575, filed Jul. 8, 1988 (now abandoned), and aContinuation-in-Part of U.S. application Ser. No. 07/397,360, filed Aug.23, 1989 (now abandoned), which is a continuation of U.S. applicationSer. No. 07/069,740, filed Jul. 6, 1987 (now abandoned).

FIELD OF THE INVENTION

The present invention relates to a column packing material useful inapplications such as blood treatment with columns and liquidchromatography used in separating and purifying proteins, enzymes,nucleic acids, monosaccharides, oligosaccharides, etc., as well as aprocess for producing such a packing material.

BACKGROUND OF THE INVENTION

Column packing materials for the applications such as liquidchromatography have conventionally been prepared from silica gel,chemically modified silica gel, polymers, carbon, etc. Porous packingmaterials are also known and have been prepared from porous silica,chemically modified porous silica, porous polymers, etc.

Calcium phosphate based compounds, in particular synthetichydroxyapatite represented by (Ca₁₀(PO₄)₆(OH)₂), have the samecomposition as the inorganic main components of teeth and bones, and,taking advantage of tis superior biocompatibility, artificial dentalroots or bone prosthetic materials made of synthetic hydroxyapatite havebeen developed. The biological affinity of hydroxyapatite has beenascribed to the close relationship to biological high polymericsubstances such as proteins and sugars.

Attempts have been made for many years to produce packing materials forliquid chromatography from such hydroxyapatite, which is closely relatedto the living body. In recent years, packing materials that arecharacterized either by the process of their production or by theirshape have been proposed, as described, for example, in UnexaminedPublished Japanese Patent Application No. 143762/1985. Packing materialsbased on hydroxyapatite have both cation and anion exchangingproperties, and work in a normal-phase mode in analysis of substancessuch as glycosides. Therefore, hydroxyapatite based packing materialsare versatile in that a single column packed with them can be used in abroad range of applications.

One problem with the prior art hydroxyapatite based packing materials,which are not fired, is that they do not have sufficiently high pressureresistance to allow for rapid passage of a mobile phase, with subsequentdifficulty in separating large volumes of a sample within a short periodof time. As a further problem, the conventional hydroxyapatite basedpacking materials are highly soluble, so if a mobile phase is caused toflow over an extended period, the surface of the packing materialdissolves to cause deterioration of its separating performance (i.e.,resolution). Furthermore, the fine particles of hydroxyapatite resultingfrom the dissolved surface of the packing material will tend to blockthe passage of the mobile phase and clog the column filter, therebyrendering it no longer usable.

Another major problem with such packing materials prepared from calciumphosphate based compounds is that it is extremely difficult to formgranules having a uniform shape and size. When such packing materialsare used in liquid chromatography, the number of theoretical platesattainable is small, tailing is prone to occur in chromatograms, anddifficulty is encountered in adjustment of the pressure for pumping amobile phase and controlling its flow rate.

Further, the conventional calcium phosphate based compounds have beenchiefly intended for separation of materials such as proteins andenzymes, and in order to attain higher resolution separations, most ofthe compounds have been used in a porous state having a large surfacearea. Porous packing materials are also used in gel permeationchromatography (see, for example, Unexamined Published Japanese PatentApplication No. 155290/1975). In gel permeation chromatography, however,materials of similar chemical composition are separated in the order oftheir molecular weight, with the higher molecular weight componentseluting first.

The present inventors have found that if a porous packing material madeof a calcium phosphate based compound is used in separation ofsaccharides, the separating performance of the packing material isadversely affected by its propensity to cause separation of componentsin the decreasing order of their molecular weight, as in the case of gelpermeation chromatography.

A further problem with calcium phosphate compounds is that theygenerally have high solubility in acidic solutions, which is a seriousproblem in various applications and conditions under which packingmaterials are intended or desired to be used. That is, hydroxyapatitepacking shows both cation exchanging ability and anion exchangingability to proteins, etc., while exhibiting high ability in separationof glycosides in the normal phase mode using acetonitrile and water asan eluent. Owing to such characteristics, a single column packed withhydroxyapatite can be applied to separation of a variety of substances.Since the desired substance can be separated under mild elutionconditions, the sample under chromatography is protected fromdeactivation. Furthermore, the column has a high recovery. Therefore,with developments in the biological industry, hydroxyapatite has beenregarded as one of the most promising packings for chromatography. Thatis, hydroxyapatite is the only one of the apatite compounds which hashitherto been used not only as an implant material, but also as apacking for liquid chromatography, as described in Journal of LiquidChromatography, Vol. 9(16), pp. 3543-3557 (1986).

However, the hydroxyapatite packing is poor in resistance to dissolutionin acidic solutions, sometimes failing to fulfill its function. That is,when an acidic mobile phase is passed through the column packed withhydroxyapatite for a long period of time, crystals of hydroxyapatite aredissolved out and fine crystals released from the surface of packingparticles and obstruct the passage of the mobile phase, eventuallybecoming useless. Therefore, the conventional hydroxyapatite packing isnot suitable for separation operation in an acidic region. Particularlyat a pH of 5.5 or less such packing cannot be used continuously and therange of substances to which it is applicable is naturally limited.

With respect to hydroxyapatite containing fluorine, fluoride uptake byhydroxyapatite has been reported, as described in Colloids and Surfaces,Vol. 13, pp. 137-144 (1985). However, its application to chromatographyhas not yet been reported or established.

SUMMARY OF THE INVENTION

An object, therefore, of the present invention is to provide a packingmaterial that comprises a calcium phosphate based compound and which isin the form of spherical granules of a uniform size.

A further object of the present invention is to provide a packingmaterial for liquid chromatography that has high resolution and exhibitssuperior resistance to pressure and dissolution.

A further object of the present invention is to provide a packingmaterial that is made from a calcium phosphate based compound and whichis suitable for use in separation of saccharides.

Another object of the present invention is to provide a packing materialfor liquid chromatography which exhibits high performance for separatinga wide range of substances and excellent resistance to dissolution andmaintains its functions stably for a long period of time.

Yet another object of the present invention is to provide a process forproducing such packing materials.

Other objects and effects of this invention will be apparent from thefollowing description.

In accordance with a first aspect of the present invention, a packingmaterial is provided comprising particles composed of a sphericalsubstrate having a coating of a calcium phosphate based compound on thesurface thereof.

A second aspect of the present invention concerns a packing material forliquid chromatography that is composed of porous calcium phosphate basedgranules having open pores with an average pore size of from 0.01 to 20μm, said granules being composed of crystalline particles with anaverage size (average diameter) of from 0.1 to 10 μm.

The second aspect of the present invention involves a process forproducing a packing material for liquid chromatography that comprisesmixing calcium phosphate based particles with pyrolyzable particleshaving an average size of from 0.02 to 30 μm, granulating the mixing,and firing the granulation at 900 to 1,400° C. to cause completecombustion of the pyrolyzable particles.

The second aspect of the present invention further involves a processfor producing a packing material for liquid chromatography thatcomprises providing a slurry containing calcium phosphate basedparticles, adding a foaming agent to the slurry to form bubbles havingan average size of from 0.02 to 30 μm, drying the foam to make a porousmaterial, grinding the porous material into granules, and firing thegranules at from 900 to 1,400° C.

In the third aspect of the present invention, the present inventors madefurther studies on the separation of components other than proteins andenzymes with packing materials made of calcium phosphate based compoundssuch as hydroxyapatite. As a result, it has now been found that certaindense calcium phosphate based compounds are effective in the separationof saccharides, with the separation pattern being such that the lowermolecular weight components elute first.

The spherical packing material for liquid chromatography of the thirdaspect of the present invention comprises spherical particles of atleast one material selected from the group consisting ofCa₁₀(PO₄)₆(OH)₂, Ca₃(PO₄)₂, Ca₂P₂O₇, Ca(PO₃)₂, Ca₁₀(PO₄)₆F₂ andCa₁₀(PO₄)₆Cl₂, and said packing material has a dense structure with aporosity of no more than 5%.

In the fourth aspect of the present invention, in order to provide apacking for liquid chromatography excellent in dissolution resistanceincluding acid resistance and yet maintaining the high separationperformance of hydroxyapatite, the present inventors have investigatedthe use of fluoroapatite (one of the compounds having the apatitestructure) as the packing and accomplished the present invention.

The fourth aspect of the present invention thus relates to a packing forliquid chromatography comprising particles having at least on thesurface thereof a fluoroapatite represented by formula (I):

Ca₁₀(PO₄)₆(OH)_(2−2x)F_(2x)  (I)

wherein x represents a number of from about 0.1 to 1, preferably fromabout 0.4 to 1, and more preferably about 1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an electron micrograph (×100) showing the structure of agranule in the packing material prepared in Example 5;

FIG. 2 is an electron micrograph showing the structure of the sameparticle, but at a higher magnification (×5,400);

FIG. 3 is an electron micrograph (×15,000) showing the structure of aparticle in the packing material prepared in Example 6;

FIG. 4 is a chromatogram showing the results of separating glucose fromsaccharose by liquid chromatography using the packing material preparedin Example 6;

FIGS. 5 and 6 are each a scanning electron micrograph showing theparticulate structure of the packing obtained in Example 7;

FIG. 7 is an X-ray diffraction pattern of the packing obtained inExample 7;

FIGS. 8(a) and 8(b) are each an infrared absorption spectrum of thepacking obtained in Example 7 and a hydroxyapatite packing,respectively;

FIG. 9(a) is a chromatogram obtained by using the packing obtained inExample 7;

FIG. 9(b) is a chromatogram obtained by using a hydroxyapatite packing;and

FIG. 10 is a chromatogram obtained by using the packing obtained inExample 8.

DETAILED DESCRIPTION OF THE INVENTION

The first aspect of the present invention will now be discussed indetail. Since the particles of packing material in the first aspect ofthe present invention are formed by coating the surface of a sphericalsubstrate with a calcium phosphate based compound, they are very closeto true spheres in shape and have a uniform size if the sphericalsubstrate used is selected from among various known beads. A packingmaterial comprising such particles can advantageously be used in liquidchromatography, producing desirable results such as an increase in thenumber of theoretical plates attainable, reduced occurrence of tailing,and easy adjustment of the pressure for pumping a mobile phase and itsflow rate.

Examples of the calcium phosphate based compound that can be used inmaking the column packing material of the first aspect of the presentinvention include Ca₁₀(PO₄)₆(OH)₂, Ca₃(PO₄)₂, Ca₂P₂O₇, Ca(PO₃)₂,Ca₁₀(PO₄)₆F₂ and Ca₁₀(PO₄)₆Cl₂. These calcium phosphate based compoundscan be synthesized by a variety of known methods, such as the wet methodin which a water-soluble phosphate salt is reacted with a water-solublecalcium salt in an aqueous solution, and the dry method in which aphosphoric compound is reacted with a calcium compound under elevatedtemperatures. Beads of materials such as polyesters, polystyrenes,polyacrylics, carbon, silica, alumina, and phosphate glass can be used athe spherical substrate in the present invention. Beads of suchmaterials are commonly available in the commercial market, and knownexamples include: polystyrene beads (e.g., “Fine Pearl” of SumitomoChemical Co., Ltd.), polyacrylic beads (e.g., “Fine Pearl” of SumitomoChemical Co., Ltd.), carbon beads (e.g., product of Moritex),silicabeads (e.g., “Nucleosil” of Nagel) and alumina beads (e.g., product ofMoritex with 99.5% Al₂O₃). The size of these beads may be appropriatelyselected in accordance with the size of the granules in the desiredpacking material.

A calcium phosphate based compound can be coated on the surface of thespherical substrate by any suitable method such as sputtering,agglomeration by spray drying, or agglomeration by rolling and tumbling.

The particles in the packing material of the present invention arepreferably adjusted to have a size (diameter) in the range of from 1 to100 μm. If their size is less than 1 μm, increased resistance to flowwill occur when the mobile phase is pumped into the packed column. Ifthe size of the particles exceeds 100 μm, they have such a decreasedsurface area that the resolution (separating performance) of the packingmaterial is prone to decrease.

The thickness of the coating of a calcium phosphate compound ispreferably within the range of from about 0.5 to about 50 μm. If thethickness of the coating is less than 0.5 μm, the coating will not havethe desired strength. If the thickness of the coating exceeds 50 μm, thespherical nature of the particles becomes difficult to maintain.

Turning now to the second aspect of the invention, it is known thatpacking materials for liquid chromatography are generally in the form ofporous granules so that they have large enough specific surface areas toachieve contact with as high volumes of samples as possible.Hydroxyapatite based packing materials can also be provided with agreater ability to separate proteins or enzymes if they are formed asporous granules. A convenient method for preparing porous hydroxyapatitegranules is spray drying, in which a hydroxyapatite slurry synthesizedby a conventional wet method is sprayed into an air stream at from 80 to250° C. to form porous granules having a size (diameter) of from 2 to150 μm, which are then fired at from 500 to 700° C., to provide apacking material having improved resolution and pressure resistance. Acolumn packed with this packing material can be satisfactorily used inliquid chromatography under commonly employed operating conditions.However, in liquid chromatography intended for industrial separation andpurification, more pressure resistance and durability are sometimesrequired in order to achieve processing of the desired sample in largervolumes.

In order that hydroxyapatite granules will have not only a greaterpressure resistance or strength, but also enhanced resistance todissolution, they may be fired at elevated temperatures to permitsufficient growth of crystalline particles. However, if the firingtemperature is simply elevated, voids between crystalline particles willbe lost and the hydroxyapatite granules change from the porous to densestate to become deteriorated in their separating performance.

Therefore, an important object of the present invention is to improvethe resistance to pressure and dissolution of porous hydroxyapatitegranules by firing them at elevated temperatures without sacrificingtheir porosity. This aspect of the present invention is hereinafterdescribed more specifically.

In addition to the hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂) already mentioned,fluoroapatite (Ca₁₀(PO₄)₆F₂), chloroapatite (Ca₁₀(PO₄)₆Cl₂), tricalciumphosphate (Ca₃(PO₄)₂) and various other kinds of known calcium phosphatebased compounds may be employed in the present invention. Such calciumphosphate based compounds can be synthesized by known wet and drymethods.

According to one method for producing the packing material of the secondaspect of the present invention, calcium phosphate based particles aremixed with pyrolyzable particles having an average size of from 0.02 to30 μm, and the mixture is granulated and fired at from 900 to 1,400° C.to cause complete combustion of the pyrolyzable particles. In apreferred case, pyrolyzable particles may be added to a slurry of acalcium phosphate based compound that has been synthesized by a wetmethod, the resulting mixture being then spray-dried. It is necessarythat the pyrolyzable particles should not undergo thermal decompositionor deterioration during spray drying and heat-resistant resins such asfluorine resins are preferably used as the materials of such pyrolyzableparticles. Other heat-resistant resins may of course be used. Thepyrolyzable particles are preferably added in amounts ranging from 30 to70 parts by weight per 100 parts by weight of the solid content of thecalcium phosphate based compound. If less than 30 parts of weight of thepyrolyzable particles are used, the intended porosity is not attained.If more than 70 parts by weight of the pyrolyzable particles are used,the porosity of the resulting granules is too high to ensuresatisfactory strength. For increasing the strength of binding betweenthe granules formed from the sprayed slurry, a binder such as polyvinylalcohol may be added to the slurry. The resulting granules are thenfired at from 900 to 1,400° C. to cause complete combustion of thepyrolyzable particles and thereby form porous granules. By adjusting theaverage size of the pyrolyzable particles to be within the range of from0.02 to 30 μm, the fired granules can be provided with pores having anaverage size of from 0.01 to 20 μm.

According to another method for producing the packing material of thesecond aspect of the present invention, a slurry containing calciumphosphate based particles is first provided, and a foaming agent isadded to the slurry to form bubbles having an average size of from 0.02to 30 μm, the foam being then dried to form a porous material which isground into granules and fired at from 900 to 1,400° C. In this method,the foam comprising the bubbles with an average size of 0.02 to 30 μmthat have been formed by addition of the foaming agent is fired toproduce open pores having an average pore size of 0.01 to 29 μm. Asuitable foaming agent is aqueous hydrogen peroxide or ovalbumin.

The above-described methods for producing the packing material of thepresent invention are characterized in that even if the firingtemperature is elevated to be within the range of from 900 to 1,400° C.,the resulting granules have open pores with an average pore size of from0.01 to 20 μm. By employing such high firing temperatures, thecrystalline particles of which the calcium phosphate based granules arecomposed can be provided with an average size in the range of from about0.1 to 10 μm.

Therefore, the packing material of the second aspect of the presentinvention comprises porous calcium phosphate based granules having openpores with an average pore size of from 0.01 to 20 μm and thecrystalline particles of which these granules are composed have anaverage size of from 0.01 to 10 μm. The packing material having thesedimensional features can be used in liquid chromatography for achievinghigh resolution while exhibiting strong resistance to pressure anddissolution. Granules having open pores with an average pore size ofless than 0.01 μm are difficult to produce. On the other hand, if theaverage pore size of open pores exceeds 20 μm, not only the specificsurface area, but also the strength of the granules will be decreased.If the crystalline particles of which the granules are composed have anaverage size of less than 0.1 μm, satisfactory resistance to dissolutionis not attainable, and if their average size exceeds 10 μm, it becomesdifficult to make porous granules. The particles in the packing materialtaken as a whole preferably have an average size of from about 1 toabout 2,000 μm.

The third aspect of the present invention will now be explained.

Since the packing material of the third aspect of the present inventionis comprised of dense spherical particles with a porosity of no morethan 5%, it has a smaller propensity to separate components according tothe elution profile of gel permeation chromatography, and hence issuitable for use in separating saccharides in the increasing order oftheir molecular weight.

The packing material of the present invention is made of at least onematerial selected from the group consisting of Ca₁₀(PO₄)₆(OH)₂,Ca₃(PO₄)₂, Ca₂P₂O₇, Ca(PO₃)₂, Ca₁₀(PO₄)₆F₂, and Ca₁₀(PO₄)₆Cl₂. Thesecalcium phosphate based compounds can be synthesized by a variety ofknown methods, such as the wet method in which a water-soluble phosphatesalt is reacted with a water-soluble calcium salt in an aqueous solutionand the dry method in which a phosphoric compound is reacted with acalcium compound under elevated temperatures.

The calcium phosphate based compounds listed above are granulated tospherical particles by various techniques such as spray drying and therolling/tumbling combination. In the method of granulation by spraydrying, a calcium phosphate based compound is dispersed in water and theresulting slurry is spray-dried at a temperature of from about 100 to250° C. to make spherical granules. In the method of granulation byrolling and tumbling, the particles of a calcium phosphate basedcompound are fed from above onto the central part of a tilted rotarydisk and are agglomerated under the rotating action of the disk.

The granules are then fired at a temperature of from 900 to 1,400° C. tomake a packing material that comprises spherical particles with aporosity of no more than 5%. If the firing temperature is less than 900°C., a packing material with a porosity exceeding 5% will often result,and the propensity of such packing material to cause separation ofcomponents in the decreasing order of their molecular weight, as in gelpermeation chromatography, makes it unsuitable for use in the separationof saccharides. If the firing temperature exceeds 1,400° C., the calciumphosphate based compound will undergo a decomposition reaction, and theresulting packing material will have a low resolution.

The particles in the packing material of the third aspect of the presentinvention need not be completely and perfectly spherical in shape,provided that they are generally spherical in shape, and shapes likeeggs or rugby balls are also included within the term “spherical” asused in association with the description of the third aspect of thepresent invention.

The average size (average diameter) of the particles in the third aspectof the present invention is not limited to any particular value, but ispreferably in the range of from about 1 to about 100 μm. If the averagesize is less than 1 μm, the packing material will have an increasedresistance to the passage of a mobile phase through a column. If, on theother hand, the average size exceeds 100 μm, the packing material willhave a decreased resolution.

The packing material of the third aspect of the present invention provesparticularly effective in the separation of monosaccharides such asglucose or oligosaccharides such as sucrose, lactose, and raffinose,producing a separation pattern in which the lower molecular weightcomponents elute first.

The fourth aspect of the present invention, directed to a packing forliquid chromatography comprising particles having at least on thesurface thereof a fluoroapatite represented by formula (I):

Ca₁₀(PO₄)₆(OH)_(2−2x)F_(2x)  (I)

wherein x is a number of from about 0.1 to 1, will now be explained.

Fluoroapatite which constitutes at least the surface of the packingparticles of the present invention includes not only pure fluoroapatitewherein the hydroxyl groups are completely substituted by a fluorineatom (fluorination degree x is 1), but also partially fluorinatedhydroxyapatite wherein only a part of the hydroxyl groups is substitutedwith a fluorine atom to a fluorination degree of at least about 0.1. Ifthe fluorination degree is less than about 0.1, sufficient improvementon acid resistance cannot be reached.

The inside structure of the individual packing particles according tothe present invention is not particularly restricted as long as thesurface thereof comprises fluoroapatite represented by formula (I).Examples of the embodiment of the invention include (1) a packingcomprising fluoroapatite of formula (I) throughout the individualparticles, (2) a packing comprising hydroxyapatite particles of whichsurface is fluorinated to have formula (I), and (3) a packing comprisinginert carrier particles coated with fluoroapatite of formula (I).

In the above embodiment (1), the whole of the individual particles isformed of the fluoroapatite of formula (I) and preferably has a specificsurface area of from about 0.01 to 20 m²/g. The porosity can becontrolled by changing the calcinating temperature or the density of theparticle forming material.

In the above embodiments (2) and (3), the thickness of the fluoroapatitesurface layer is preferably about 1 μm or more. The specific surfacearea of the packing is preferably from about 0.01 to 20 m²/g.

The embodiment (1) can be prepared by the method described, e.g., inColloids and Surfaces, Vol. 13, pp. 137-144 (1985). The embodiment (2)can be prepared by reacting a hydroxyapatite packing with a solutioncontaining fluoride ions and then calcinating the same. The embodiment(3) can be prepared by coating fluoroapatite on a carrier, e.g., bysputtering, ion-plating and thermal-spraying.

The packing comprising fluoroapatite throughout the particle accordingto the first embodiment can be obtained by known processes for producingfluoroapatite such as the method as described in Napper, D. H., Synthe,B. H.: “The Dissolution Kinetics of Hydroxyapatite in the Presence ofKink Poisons”, J. Dent. Res., Vol. 45, pp. 1775-1783 (1966). Whether noCaF₂ has been formed can be confirmed by calcining the resultingfluoroapatite at an appropriate temperature and subjecting it to X-raydiffractometry. Formation of fluoroapatite can be confirmed by the shiftof the (300 ) peak to a higher angle side by the method described, e.g.,in Moreno, E. C., Kresak, M. Zahradnik, R. T.: “Physicochemical Aspectsof Fluoride-Apatite Systems Relevant to the Study of Dental Caries”,Caries Res., Vol. 11 (Suppl. 1), pp. 142-171 (1977).

The packing comprising hydroxyapatite particles of which surface isfluorinated according to the second embodiment can be prepared, forexample, by treating the surface of hydroxyapatite particles withhydrogen fluoride under a controlled pH condition.

The packing comprising inert carrier particles (e.g., alumina) coatedwith fluoroapatite according to the third embodiment can be prepared,for example, by sputtering.

The packing particles for liquid chromatography of the fourth aspect ofthe present invention are not particularly limited in size, shape,porosity, etc. However, performances such as separating ability can beassured by following a general particle design for packings for liquidchromatography. For example, the packing preferably has an averageparticle diameter of from about 2 to 100 μm, more particularly fromabout 10 to 100 μm for industrial use and from about 2 to 10 μm for usein analyses. If the average particle size is less than about 2 μm, thepressure loss on passing a liquid sample through a column packed withthe packing becomes too large. If it exceeds about 100 μm, the surfacearea of the packing per unit volume is too small to assure separatingability. The packing preferably has a shape near to a spherical form inorder to obtain stable separation characteristics while preventingcracks or cutouts although those having a macadamized form may be used.The porosity is preferably high in view of the load of the samples, butnon-porous packing may be used for the analytical use. The specificsurface area is preferably from about 0.01 to 20 m²/g although dependingon the form of the packing particles.

The packing of the present invention can be used for a method for liquidchromatography by (a) packing a column with the packing of the presentinvention, (b) contacting the packing with a sample comprising at leastone solute, and (c) contacting the packing with a liquid mobile phase toseparate the solute by elution.

Upon carrying out the method for liquid chromatography using the packingmaterial in accordance with the fourth aspect of the present invention,the preferred eluents are as follows: In an ion exchanging mode, (1) asodium phosphate buffer (pH 5 to 9), (2) a potassium phosphate buffer,(3) a mixture of a sodium chloride solution and various buffers (e.g.,tris buffer, pipes buffer, etc.), and (4) a mixture of a potassiumchloride solution and various buffers (e.g., tris buffer, pipes buffer,etc.). In the cases of (1) and (2), a gradient elution at aconcentration of from 1-10 to 400 mM is preferred and in the cases of(3) and (4), a gradient elution at a concentration of from 10-100 mM to1 M is preferred. In a normal mode, an isocratactic elution with theacetonitrile/water ratio of from about 7/3 to 9/1 and a gradient elutionwhile increasing the water concentration are preferred.

The packing for liquid chromatography according to the present inventioncan be suitably applied to separation of solutes such as proteins (e.g.,monoclonal antibody and fibronectin), enzymes (e.g., ligase andprotease), nucleic acids (e.g., nucleotide, oligonucleotide, DNA andRNA), glycosides (ginsenoside, steviside, rebaudioside and saponin), andso on and exhibits stale separation performance even in an acidicsolution, e.g., phosphoric aid, hydrochloric acid, etc.

Upon carrying out chromatography using an open column, solutes areusually firstly in contact with the packing and a liquid mobile phase isthen made to flow through the column. On the other hand, in the caseusing HPLC (high performance liquid chromatography) as in the followingExamples, solutes are contracted with the packing simultaneously withthe liquid mobile phase in the dissolved state. That is, a solution of aliquid mobile phase containing solutes is passed through the column.

The present invention is now illustrated in greater detail withreference to the following Examples and Comparative Examples, but thepresent invention is not to be construed as being limited thereto.

Unless otherwise indicated, all parts, percents, ratios and the like areby weight.

EXAMPLES Example 1 (First Embodiment)

Silica beads (“Nucleosil” of Nagel; particle size 30 μm) were used as aspherical substrate. Hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂) was used as acalcium phosphate based compound. Model “SPF210H RF” (product of NipponElectric Anelva Co., Ltd.) was used as sputtering apparatus.

The silica beads were placed in the coating substrate holder which wasequipped with a rotating mechanism to allow for the coating of theentire surface of the beads. The hydroxyapatite was set in the targetvessel.

With proper settings of operating parameters such as the distancebetween the target and the coating substrate, high-frequency power forsputtering, and sputtering pressure, presputtering was conducted for aperiod of from 10 to 30 minutes as to clean the surface of the target.

Following the presputtering, RF (radio frequency) sputtering wasconducted for 4 hours under the following conditions:target-to-substrate distance, 40 mm; sputtering pressure, 5.5×10 Pa;sputtering power, 100 W; and sputter rate, 40 Å/min. The resultingsample was heat-treated at 700° C. for 1 hour in an argon atmosphere.Analysis with an X-ray diffractometer and a spectrophotometer revealedthat the coating on the substrate particles in the sample was made ofhydroxyapatite and had a thickness of 1.2 μm.

The packing material obtained by the above procedure was composed ofnearly spherical 32.4 μm in diameter, with a hydroxyapatite coating 1.2μm thick. This packing material was packed in a wet (swollen) state intoa stainless steel of column 7.5 mm in diameter and 100 mm long; usingthis column, standard proteins were analyzed by high-performancechromatography (chromatograph, Shimadzu LC-6A). The conditions andresults of the analysis are shown below:

Mobile phase: sodium phosphate buffer solution (pH 6.8) with a lineargradient of 0.01-0.4 M over 30 min.

Flow rate: 1 ml/min.

Pressure: 20 kg/cm²

Samples: BSA, lysozyme, and cytochrome C

Number of apparent theoretical plates: 15,000 (based on lysozyme peak)

Tailing: Small

Example 2 (First Embodiment)

Silica beads (“Nucleosil” of Nagel; particle size 30 μm) were used as aspherical substrate and hydroxyapatite were used as a calcium phosphatebased compound.

A slurry was prepared by dispersing the hydroxyapatite in water at aconcentration of 1%. The silica beads were charged into the slurry andstirred well. With continued stirring, the slurry was spray-dried with aMobile Minor type spray drier (Ashizawa-Niro) at an outlet temperatureof 90 to 110° C. to form granules comprising silica beads with a surfacecoating of hydroxyapatite. The granules were heat-treated at 700° C. for1 hour in an argon atmosphere to make a packing material, which wascomposed of particles 32 μm in diameter, with a hydroxyapatite coating 1μm thick. Using this packing material, standard proteins were analyzedas in Example 1. The conditions and results of the analysis are shownbelow:

Mobile phase: sodium phosphate buffer solution (pH 6.8) with a lineargradient of 0.01-0.4 M over 30 min.

Flow rate: 1 ml/min.

Pressure: 20 kg/cm²

Samples: BSA, lysozyme, and cytochrome C

Number of apparent theoretical plates: 14,000 (based on lysozyme peak)

Tailing: Small

Example 3 (First Embodiment)

Silica beads (“Nucleosil” of Nagel; particle size, 30 μm) were used as aspherical substrate. Hydroxyapatite was used as a calcium phosphatecompound.

A mixture of the silica beads and hydroxyapatite were charged into arolling-tumbling agglomerator and a hydroxyapatite coating was formed onthe surface of each silica bead. The coated silica beads wereheat-treated at 700° C. for 1 hour in an argon atmosphere to form apacking material, which was composed of particles 35.2 μm in diameter,with a hydroxyapatite coating 2.6 μm thick. Using this packing material,standard proteins were analyzed as in Example 1. The conditions andresults of the analysis are shown below:

Mobile phase: sodium phosphate buffer solution (pH, 6.8) with a lineargradient of 0.01-0.4 M over 30 min.

Flow rate: 1 ml/min.

Pressure: 20 kg/cm²

Samples: BSA, lysozyme, and cytochrome C

Number of apparent theoretical plates: 13,000 (based on lysozyme peak)

Tailing: Small

Comparative Example 1 (First Embodiment)

A slurry of 1% hydroxyapatite dispersed in water was spray-dried with aMobile Minor type spray drier (Ashizawa-Niro) at an outlet temperatureof 90 to 110° C. to form hydroxyapatite granules. The granules were thenheat-treated at 700° C. for 1 hour in an argon atmosphere to make apacking material, which was an agglomeration of granules from 2 to 20 μmin size (average size, 3.5 μm). Using this packing material, standardproteins were analyzed as in Example 1. The conditions and results ofthe analysis are shown below:

Mobile phase: sodium phosphate buffer solution (pH 6.8) with a lineargradient of 0.01-0.4 M over 30 min.

Flow rate: 1 ml/min.

Pressure: 80 kg/cm²

Samples: BSA, lysozyme, and cytochrome C

Number of apparent theoretical plates: 4,000 (based on lysozyme peak)

Tailing: Large

As described in the foregoing, the particles in the packing material ofthe present invention are formed by coating the surface of a sphericalsubstrate with a calcium phosphate based compound, so they are veryclose to true spheres in shape and have a uniform size. The packingmaterial comprising these particles can advantageously be used in liquidchromatography, producing such results as the increase in the number oftheoretical stages attainable, reduced occurrence of tailing, and easyadjustment of the pressure for pumping a mobile phase and its flow rate.

Example 4 (Second Embodiment)

A hundred grams of tricalcium phosphate (Wako Pure Chemical Industries,Ltd.) were ground in a dry ball mill for 24 hours to make particles. Theball mill was then charged with 10 liters of water and 40 g of fluorineresin beads “Fulolon No. 1” (trademark for product of Nippon JunkatsuzaiRyutsu Center; average bead size, 0.02 μm) and the contents were stirredfor 24 hours to obtain a uniform slurry. With continued stirring, theslurry was spray-dried with a Mobile Minor type spray drier(Ashizawa-Niro) at an outlet temperature of from 90 to 110° C. to formgranules of from 2 to 20 μm in size.

The granules were then fired in an electric furnace under the followingconditions: heating up to 700° C. at a rate of 5° C./h; subsequentheating from 700° C. to 1,100° C. at a rate of 100° C./h; holding at1,100° C. for 3 hours; and cooling to room temperature at a rate of 200°C./h. The resulting porous granules had open pores with an average poresize of 0.01 μm, with crystalline tricalcium phosphate particles havingan average size of 0.7 μm and a specific surface area of 3.46 m²/g.

The porous granules were packed in a wet (swollen) state into astainless steel column 7.5 mm in diameter and 100 mm long; using thiscolumn, standard proteins were analyzed by high-performance liquidchromatography (chromatograph, LC-6A of Shimadzu Seisakusho, Ltd.). Theconditions and results of the analysis are shown below:

Mobile phase: sodium phosphate buffer solution (pH, 6.8) with a lineargradient of 0.01-0.4 M over 30 min.

Flow rate: 1 ml/min.

Pressure: 15 kg/cm²

Samples: BSA, lysozyme, and cytochrome C

Number of apparent theoretical plates: 12,000 (based on lysozyme peak)

Durability: ≧500 cycles

Example 5 (Second Embodiment)

A hundred grams of tricalcium phosphate (Wako Pure Chemical Industries,Ltd.) were put into a polyethylene bag. After charging 185 g of anaqueous solution of 0.5% H₂O₂ into the bag, the contents were wellkneaded by hand. The resulting slurry was transferred into aheat-resistant container which was placed in a drier with internal aircirculation for 24 hours at 100° C. to obtain a dried foam. The foam wasdissipated by hand and passed through a nylon sieve to obtain porousgranules of from 10 to 20 μm in size.

The granules were then fired in an electric furnace under the followingconditions: heating up to 700° C. at a rate of 5° C./h; subsequentheating from 700° C. to 1,200° C. at a rate of 100° C./h; holding at1,200° C. for 3 hours; and cooling to room temperature at a rate of 200°C./h. The resulting porous granules had open pores with an average poresize of 3 μm, with crystalline tricalcium phosphate particles having anaverage size of 2 μm and a specific surface area of 0.52 m²/g. Electronmicrographs of these porous granules are reproduced in FIG. 1 (×100) andFIG. 2 (×5,400).

A packing material composed of these porous granules was packed in a wet(swollen) state into a stainless steel column 7.5 mm in diameter and 100mm long; using this column, standard proteins were analyzed byhigh-performance liquid chromatography (chromatograph, LC-6A of ShimadzuSeisakusho, Ltd.). The conditions and results of the analysis are shownbelow:

Mobile phase: sodium phosphate buffer solution (pH, 6.8) with a lineargradient of 0.01-0.4 M over 30 min.

Flow rate: 1 ml/min.

Pressure: 16 kg/cm²

Samples: BSA, lysozyme, and cytochrome C

Number of apparent theoretical plates: 11,000 (based on lysozyme peak)

Durability: ≧500 cycles

The results of Examples 4 and 5 show that the packing material preparedin accordance with the present invention has high resolution andexhibits superior pressure resistance and durability.

As described in the foregoing, the packing material of the presentinvention is composed of porous calcium phosphate based granules havingopen cells with an average pore size of from 0.01 to 20 μm, and thecrystalline particles of which the granules are composed have an averagesize of from 0.1 to 10 μm. Because of these dimensional features, thepacking material has high resolution and exhibits superior pressureresistance and durability. According to the process for producing thispacking material, calcium phosphate based particles are mixed withpyrolyzable particles having an average size of from 0.02 to 30 μm, or aslurry containing calcium phosphate based particles is mixed with afoaming agent to form bubbles having an average size of from 0.02 to 30μm. In either method, the resulting granules can be fired at from 900 to1,400° C. wihtout forming a solidified structure, and instead, the firedgranules have open pores with an average pore size of from 0.01 to 20μm. By selecting a firing temperature within the above-specified range,the crystalline particles of which the granules are composed can beadjusted to have an average size of from 0.1 to 10 μm, which serves toimpart superior pressure resistance and durability to the porousgranules.

Example 6 (Third Embodiment)

A solution of a phosphate salt and a solution of a calcium salt werereacted by a known method to form a hydroxyapatite slurry. The slurrywas granulated by spray drying with a Mobile Minor type spray drier(Ashizawa-Niro). The resulting granules, nearly spherical in shape, werefired at 1,100° C. for 4 hours to obtain a packing material of the typeintended by the present invention. The particles in this packingmaterial had an average size of 5 μm and a porosity of 1%. An electronmicrograph (×15,000) of a single particle in this packing material isshown in FIG. 3.

The packing material was packed into a stainless steel column (7.5mm^(φ)×100 mm^(L)) for high-performance liquid chromatography, andseparation between glucose and saccharose was effected on this columnwith an aqueous solution of 80% acetonitrile being supplied as an eluentat a flow rate of 1.5 ml/min. The chromatogram obtained is shown in FIG.4, in which the vertical axis represents absorbance at 195 nm and thehorizontal axis, time in minutes. In FIG. 4, G is a glucose peak and S asaccharose peak. As FIG. 4 shows, the packing material of the presentinvention is capable of separating saccharides at high resolution.

As described in the foregoing, the packing material of the presentinvention is generally composed of nearly spherical particles of acalcium phosphate based compound that have a dense structure with aporosity of no more than 5%. This packing material has a decreasedpropensity to cause separation of components in the decreasing order oftheir molecular weight such as occurs in the case of gel permeationchromatography, and hence is suitable for use in separation ofsaccharides in order of their molecular weight with the lower molecularweight components eluting first.

Example 7 and Comparative Example 2 (Fourth Embodiment)

Fluoroapatite was synthesized by a conventional wet process as describedin Colloid and Surfaces, Vol. 13, pp. 137-144 (1985). The resultingslurry was spray-dried by means of a spray drier (“Mobile Minor Model”manufactured by Ashizawaniro Co., Ltd.) to obtain packing particleshaving an average particle diameter of 10 μm. FIG. 5 is a scanningelectron micrograph 1,000×magnification) showing the shape of theresulting packing particles. FIG. 6 is a scanning electron micrograph(10,000×magnification) showing the surface of the packing particles.

A part of the packing was calcined at 1,100° C. and subjected to X-raydiffractometry. The results obtained are shown in Table 1 below and FIG.7. As is shown in the X-ray diffraction pattern of FIG. 7, it wasconfirmed that no CaF₂ had been formed. Further, the d value at the(300) peak was compared with the corresponding ASTM measured value asshown in Table 1 to confirm the fluorination degree. Furthermore, thepacking was analyzed by infrared absorption and the results obtained areshown in FIG. 8(a). On comparison with the infrared absorption spectrumor hydroxyapatite as shown in FIG. 8(b), it was seen that the packinghad no absorption due to the hydroxyl group in the vicinity of 660 cm⁻¹.In addition, chemical analysis of the packing revealed that the packinghad a fluorine uptake of 3.8%, which means stoichiometrical formation ofFluoroapatite. From all these considerations, the produced fluoroapatitecan be estimated to have a fluorination degree of 1.

TABLE 1 X-Ray Diffraction Pattern (211) (112) (300) a-Axis c-Axis FoundValue: Example 7 2.800 2.773 2.703 9.363 6.887 Comparative 2.816 2.7802.720 9.426 6.904 Example 2 ASTM Value: Fluoroapatite 2.80 2.776 2.7069.3684 6.8841 Hydroxylapatite 2.82 2.784 2.726 9.418 6.884

0.5 g of the resulting fluoroapatite particles was immersed in 100 ml ofan acetic acid buffer solution at a pH of 4.0 or 5.0 for 1 or 40 hours,respectively. The supernatant liquor was then filtered through aquantitative filter paper to prepare a sample solution. The samplesolution was appropriately diluted and subjected to atomic-absorptionspectroscopy to determine a Ca concentration.

For comparison, hydroxyapatite packing particles (Comparative Example 2)obtained by a conventional synthesizing method followed by spray-dryingin the same manner as described above were immersed in an acetic acidbuffer solution to prepare a sample solution in the same manner asdescribed above. The resulting sample solution was appropriately dilutedand subjected to atomic-absorption spectroscopy to determine its Caconcentration. The results obtained are shown in Table 2 below.

TABLE 2 Acid Resistance Test Ca Concentration (ppm) pH = 4.0, 1 Hr. pH =5.0, 40 Hrs. Example 7 205 68 Comparative 805 383 Example 2

As is apparent from Table 2, the amount of calcium dissolved out of thefluoroapatite of Example 7 is decreased to ¼ in the case of short-termimmersion and ⅕ in the case of long-term immersion as compared with thatof hydroxyapatite of Comparative Example 2, indicating superiority offluoroapatite in acid resistance.

Then, each of the packings of Example 7 and Comparative Example 2 wasfilled by a wet process in a stainless steel column having a diameter of7.5 mm and a height of 100 mm, and the packed column was set in a liquidchromatograph “LC-6A” manufactured by Shimazu Seisakusho Co., Ltd.). Asample solution containing a phosphoric acid buffer solution (pH=6.8)having dissolved therein 10 μg/μl of bovine serum albumin (produced bySeikagaku Kogyo Co., Ltd.), 1.25 μg/μl of lysozyme (produced bySeikagaku Kogyo Co., Ltd.; prepared from egg white by repeatingcrystallization 6 times), and 5 μg/μl of cytochrome C (prepared fromhorse heart; manufactured by Shiguma Co., Ltd.) was passed through thecolumn at a flow rate of 1.0 ml/min to obtain a chromatogram. Theresults obtained are shown in FIGS. 9(a) and (b). As is apparent fromFIGS. 9(a) and 9(b), the fluoroapatite of Example 7 {FIG. 9(a)} provedcapable of obtaining a separation pattern similar to that ofhydroxyapatite of Comparative Example 2 {FIG. 9(b)}.

Example 8 (Fourth Embodiment)

A hydroxyapatite slurry was synthesized by a conventional wet processusing a calcium hydroxide slurry and a phosphoric acid aqueous solution.The resulting hydroxyapatite slurry was spray-dried by means of a spraydryer (“OC-20” made by Ohkawara Kakouki Co., Ltd.) to obtainhydroxyapatite particles. After calcined at 700° C., 50 g of theparticles were suspended into 150 g of 2.7% sodium fluoride aqueoussolution. The resulting slurry was filtrated, dried and calcined at 700°C. to obtain a packing material. The resulting packing material had anaverage diameter of about 20 μm and a porosity of about 60%.

The packing material obtained above was filled by a wet process in astainless steel column having a diameter of 7.5 mm and a height of 100mm, and the packed column was set in a liquid chromatograph (“LC-6A”manufactured by Shimazu Seisakusho Co., Ltd.). A sample solutioncontaining a sodium phosphate buffer (pH: 6.8) having dissolved thereinbovine serum albumin, lysozyme and cytochrome C with a linear gradientof 0.01 to 0.4 M over 30 minutes and retained at 0.4 M for 15 minuteswas passed through the column at a flow rate of 1 ml/min and a pressureof 8 kgf/cm² to obtain a chromatogram. The resulting chromatogram isshown in FIG. 10 attached. The number of theoretical plates for thelysozyme peak was 300.

As described above, since the packing for liquid chromatography inaccordance with the present invention comprises fluoroapatite in atleast its surface, it was excellent in resistance to dissolution andexhibits high and stable performance in separation of a broad range ofsubstances. Therefore, the packing of the present invention can be usedadvantageously for separation and purification of proteins, enzymes,nucleic acid, and the like.

While the invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to one skilled in theart that various changes and modifications can be made therein withoutdeparting from the spirit and scope thereof.

What is claimed is:
 1. A packing for liquid chromatography comprisingporous calcium phosphate based granules having open pores with anaverage pore size of from 0.01 to 20 μm, said granules being composed ofcrystalline particles with an average size of from 2 to 10 μm.